|Publication number||US5804450 A|
|Application number||US 08/604,083|
|Publication date||Sep 8, 1998|
|Filing date||Feb 20, 1996|
|Priority date||Feb 21, 1995|
|Also published as||CA2170003A1, DE19505894A1, EP0729032A2, EP0729032A3, EP0729032B1|
|Publication number||08604083, 604083, US 5804450 A, US 5804450A, US-A-5804450, US5804450 A, US5804450A|
|Original Assignee||Boehringer Mannheim Gmbh|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (3), Referenced by (19), Classifications (11), Legal Events (7)|
|External Links: USPTO, USPTO Assignment, Espacenet|
The invention addresses a method for the specific determination of LDL in biological liquids in the presence of an LDL precipitating agent and/or a water-soluble polymer, especially a polyanion and a detergent and/or a detergent mixture. The invention also relates to a reagent suitable therefor which brings about the advantage of a significantly reduced VLDL interference. The polymers are in particular those with a branched structure, where the side branches have acid groups, especially branched alkane sulphonic groups.
The determination of the LDL level in plasma and especially in serum has gained high clinical importance: up to the 80% of the total cholesterol contents are transported in the form of so-called low-density lipoproteins (LDL or β-fraction) and are thus part of those particles which today are considered most significant atherogenic components in the lipid metabolism. Moreover, it is known that elevated plasma LDL concentrations can have a direct endothelial damaging effect which is comparable to other noxae such as hypertension, hyperinsulinemia or endotoxins. It is also known that the cholesterol deposited in an atherosclerotic plaque was originally also present in the plasma in the form of LDL cholesterol.
Today, there are several methods available for the quantitative determination of LDL or β-cholesterol in plasma and especially in serum (Mills, G. L., Lane, P. A., Weech, P. K.: A guidebook to lipoprotein technique. Elsevier, Amsterdam, 1984; Cremer, P. and Seidel, D.: Dtsch. Gesell. Klin. Chem. Mittl. 21, 1990, 215-232).
An essential difference is made between differentiating techniques such as ultracentrifugation and electrophoresis, on the one hand, and precipitation techniques on the other hand. The former require expensive instrumentation and are very time-consuming. They are therefore primarily used as reference and confirmation tests. Precipitation with high-molecular, mostly negatively charged substances is today a routine method in the laboratory. Dextrane sulfate, polyvinyl sulfate, polycyclic surface-active anions or heparin are used in particular as LDL precipitation reagents in today's clinical diagnostics. However, these methods have one disadvantage. The precipitation with heparin in an acid medium or with polycyclic anions in serum samples with preceding or existing lipolytic activity leads to falsely elevated results for LDL cholesterol. When using dextrane sulfate or polyvinyl sulfate (PVS), which today are considered the reagents of choice as they show the highest precision for LDL determinations, the values found in serum samples with preceding or existing lipolytic activity are falsely depressed. Moreover, dextrane sulfate-containing precipitation reagents also lead to falsely depressed results when high levels of free fatty acids or triglycerides are present in the sample to be assayed. Interference in triglyceride-rich samples is primarily caused by the presence of high VLDL concentrations. This is even more so the case in patients under heparin therapy. Another disadvantage that is common to all precipitation methods is that in addition to LDL, the LDL-like lipoprotein Lp(a) is also subject to precipitation. The methods are, hence, not entirely LDL-specific.
DE 42 23 355 describes a method for the specific determination of LDL using polymers having certain alkane sulphonic acid groups. However, this method only partially satisfies today's demands, particularly with respect to samples with a high VLDL percentage.
It was, hence, an object of the invention to provide a method for the specific determination of LDL that is easy and fast and does not exhibit interference by lipoprotein particles, particularly VLDL or increased concentrations of triglycerides or free fatty acids or heparinized samples.
This object is accomplished in that an LDL-precipitating agent or a water-soluble polymer, for example, one containing anionic side branches and a detergent or a detergent mixture is added to the samples to be assayed. Subsequently, the contents of the forming LDL-specific aggregate (agglutinate) is directly determined in a turbidimetric measurement. The VLDL interference can also be reduced in conventional routine methods with dextrane or PVS as a precipitating agent in that the detergent or detergent mixture in accordance with the invention is added.
Possible water-soluble polymers are those having anionic side groups according to DE 42 23 355 which are derived from acrylic acid ester monomers, but it is also possible to use dextrane sulfate and heparin. Particularly preferred are compounds of the general formula (I): ##STR1## wherein R1 is hydrogen or a lower hydrocarbon residue, preferably a methyl group, X is an oxygen atom or an NH group, A is a linear branched hydrocarbon chain consisting of 2 to 10 C atoms, with branched chains being preferred, and Z is a ##STR2##
Homopolymers of 2-acrylamido-2-methyl-1-propane sulphonic acid, 2-acrylic amidoglycollic acid and/or copolymers of said monomers have proven to be particularly suitable.
The polymers of the anionic side chains have a preferred molecular weight of 2×104 to 5×106 Dalton, particularly preferred about 5×105 Dalton (gel permeation chromatography).
These anionic polymers can be prepared by means of methods that are known to the expert, including solution, suspension or emulsion polymerization (Meth. d. Organ. Chemie (Houben-Weyl), Vol. E 20, Makrom. Stofte, Thieme Verlag, Stuttgart, 1987). Solvent polymerization as described in Polymer 31 (1990), 1269-1267 (Huglin and Rego) is particularly preferred. The polymerization can be accelerated with the aid of conventional initiators, especially peroxides, persulfates or persulfates/bisulfits or the corresponding azo compounds that are present in aqueous systems in the form of free radicals. This is also known to the expert.
The concentration of one or several of the branched polyanions ranges between 0.001 and 5.0 mg/ml and preferably ranges between 0.005 and 1.0 mg/ml. A particularly suitable range is one between 0.01 and 0.1 mg/ml.
The detergents to be added in accordance with the invention are particularly those with a zwitterionic character of the general formula (II): ##STR3## wherein x is a number between 1 and 20, and y is a number between 0 and 8, R1 is hydrogen, a cholamido group or a mono- or diglyceride group bound via phosphate residue,
R2 is hydrogen, a hydroxyl group or an oxyanion, an acid residue, a hydroxyalkyl sulphonic acid group or a carboxyl group.
Preferred detergents are those where x is an odd number between 11 and 17, y is a number between 1 and 3 and/or R2 is a sulphonic acid residue, a hydroxyethyl sulphonic acid residue, a hydroxypropyl sulphonic acid residue or a carboxyl residue, or R1 is hydrogen, a steroid frame, a lecithin or a lysolecithin residue.
Particularly suitable detergents are the following: N-dodecyl-N,N-dimethylammonio-3-propane sulfate or the corresponding N-tetradecyl or N-hexadecyl compound (type "Zwittergent": Zwittergent 3-14, 3-16), N-dodecyl-N,N-dimethyl-glycine (Empigen BB®), aminoxide, CHAPS, CHAPSO and α-lecithin (α-phosphatidylcholin) or α-lysolecithin (α-lysophosphatidylcholin). All detergents are commercially available.
The zwitterionic detergents are used either solely or in combination with a non-ionic detergent such as Pluronic F68 and/or Tween 20.
Generally, it is possible to use buffer substances for the method of determination of the invention provided their buffering capacity is in weakly acidic, neutral, or weakly alkaline pH range. Particularly suitable buffer substances are sodium acetate and so-called Good buffers, such as TRIS.HCl, BIS-TRIS methane, MES or imidazole. The pH value for the determination when using these buffers, preferably ranges between pH 5 and 9, particularly preferred between 7.0 and 8.0, a particularly suitable range is one between 7.3 and 7.7. The concentration of the buffer should range between 0.001 and 0.2 mol/l. A particularly suitable concentration range is one between 0.005 and 0.07 mol/l.
A preferred embodiment employs a TRIS.HCl buffer, a bis 2-hydroxyethyl!imino-tris hydroxymethyl!methane (bis-TRIS) buffer, an MES or an imidazole buffer in the presence of 2-valent metal ions. The concentration of the buffer ranges between 0.001 and 0.2 mol/l, the concentration of the metal salts between 0.001 and 0.20 mol/l and the pH value ranges between 5.5 and 9.0. In particularly preferred embodiment, the concentration of the buffer ranges between 0.005 and 0.07 mol/l, the concentration of the metal salts between 0.01 and 0.03 mol/l, and the pH value ranges between 7.0 and 8.0. Generally, it is possible to employ all salts of known 2-valent metal ions. Particularly preferred are Mg2+, Ca2+, Mn2+, and Cu2+. Mg2+ and Ca2+ have proven to be particularly suitable. The polyanion concentration should be selected as indicted above.
Another subject matter of the invention is a reagent containing a water-soluble polymer and a zwitterionic and/or a non-ionic detergent and a substance buffering in a pH range between 5 and 9. The amount of polymers used ranges between approximately 0.005 and 1.0 mg/ml of reagent solution; particularly preferred are poly-(2-acrylamido-2-methyl-1-propane sulphonic acid) (PAMPS), polyacrylic acid-(2-phospho-1,1-dimethyl-ethylamide) (PAP), poly-2-acrylamido-glycollic acid (PAAG), poly-(2-acrylamido-2-methyl-1-propane sulphonic acid-CO-2-acrylamido-glycollic acid) P(AMPS-AAG)! and/or corresponding polymers and/or corresponding poly(meth)acrylic acidic ester or even dextrane sulfate, polyvinyl sulfate or heparin in a concentration range between 0.02 and 0.04 mg/ml, particularly preferred in a concentration range of 0.02 to 0.04 mg/ml.
The amount of detergent to be added depends on the type of detergent used. The efficiency of the VLDL interference reduction in case of detergents with a shorter chain length (x, y<4) is achieved only when higher concentrations are used. The addition of a zwitterionic detergent at a concentration of approximately 0.002 to 1.0% (w/v) has proven to be particularly suitable in the method.
Particularly suitable buffer substances are sodium acetate, TRIS.HCl, BIS-TRIS, MES, and imidazole. The preferred concentration of the buffer ranges between 0.001 and 0.2 mol/l.
The given concentrations are final concentrations, i.e. the concentration found in the reaction mixture.
The pH value range of the reagent for the determination of LDL is between 5.0 and 9.0, preferably between pH 7.0 and 8.0 or between pH 7.3 and 7.7. The temperature for the determination may vary between 10° and 40° C., preferred is a temperature of approximately 37° C.
The ratio of sample volume to be assayed to reagent solution is variable. Ratios between 1:6 and 1:100 have proven to be suitable. It is preferred to mix approximately 5 parts of the serum sample (e.g. 5 μl) with 300 parts reagent solution (e.g. 300 μl). Samples are in particular biological liquids such as serum, plasma, or urine.
Moreover, it has proven to be advantageous to first prepare two separate reagent solutions; one containing a 2-valent metal ion and a second a water-soluble polymer aggregation agent. A zwitterionic and/or a non-ionic detergent is added either to the first reagent to which then the sample is added or to both reagents.
As compared to other known methods, the LDL-specific aggregation or agglutination that was triggered with an LDL precipitating agent such as dextrane sulfate, polyvinyl sulfate, or heparin and preferably with an anionic polymer (PAMPS) and one or several of the detergents in accordance with the invention has the following advantages:
1. In addition to LDL, the apoprotein B-containing lipoproteins such as very-low density lipoproteins (VLDL), Lp(a) and/or chylomicrons do not interact with the combination in accordance with the invention either, and are thus not measured.
2. High triglyceride concentrations do not negatively affect the result.
3. The determination is carried out rapidly, without sample pretreatment (approximately 1 to 10 min).
4. Forming aggregate is present in a very stable condition and can be directly measured and reproduced.
5. The determination of LDL cholesterol is linear over a very broad measuring range (50 to 350 mg/dl LDL cholesterol).
The method and/or reagent of the invention leads to an absolutely specific agglutination of LDL particles within 1 to 5 min. There is no interference by increased triglyceride concentrations, and a preceding separation of other lipoprotein fractions is not required either. The assay can hence be run on analyzers or simple photometers (turbidimetric) for the determination of the LDL concentration or the LDL cholesterol which is desirable for routine work. Moreover, it is also possible to determine the apoprotein B-100 and/or other molecular components of the LDL particle contained in the LDL agglutinate.
The broad measuring range where the method of the invention exhibits a linear measuring signal covers the diagnostically relevant range, especially the one of elevated LDL values (>190 mg/dl) with the advantage of requiring only one-point calibration. Because of the elevated linear range, it is also possible to have a more precise quantitative monitoring of elevated LDL levels without requiring expensive confirmation procedures such as lipoprotein electrophoresis or ultracentrifugation.
The method and reagent in accordance with the invention agglutinates LDL but not the additional LDL-similar Lp(a). This finding is surprising as all known LDL precipitation reagents also precipitate at least partially the Lp(a) fraction. It is, hence, possible with the present invention to also determine the Lp(a) fraction in a simple manner by reductively cleaving of the Lp(a) portion of the Lp(a) particle which is bound via a disulfide bridge subsequent to the LDL agglutination and determination in accordance with the invention. By obtaining the difference, it is possible to determine the contents of lipoprotein(a).
Moreover, note that despite the present teaching that the addition of detergents greatly affects the LDL precipitation, it was surprisingly possible to prevent the simultaneous precipitation of VLDL by adding zwitterionic detergents especially Zwittergent or mixtures of non-ionic detergents without affecting the specific precipitation of LDL. It is particularly critical to determine the optimal concentration of the detergent in the reaction mixture. If a concentration is used that is too low, the reduction of the interference is almost non-existent, while too high detergent concentrations prevent the simultaneous precipitation of VLDL but also reduce the complete precipitation of the LDL particles. With the method and reagent of the invention, it is possible to have an interference-free LDL determination up to approximately 130 mg/dl VLDL contents. This corresponds to a triglyceride contents of approximately 800 mg/dl, while routine methods such as dextrane sulfate precipitation already shows significant interference beginning at 400 mg/dl of triglycerides.
It is also possible to carry the method of the invention out on test strips or similar matrices, provided the individual reaction components are impregnated on or in a carrier material or covalently bound. Possible carrier material is absorbent or film-forming carrier material with the capability to swell as it is commonly used for test strips, e.g. paper or similar fleece material such as tea bag paper. The reaction components can be distributed on several carriers that are in contact with one another or serve as carriers themselves. Covalently bound polyanions (e.g. PAMPS) allow the complete, selective separation of LDL on a test strip. The LDL cholesterol value can be determined by measuring the cholesterol contents of the non-bound lipoprotein particles and obtaining the difference with total cholesterol contents.
CHAPS= 3-(3-cholanidopropyl)-dimethylammonio!-1-propane sulfate
CHAPSO= 3-(3-cholanidopropyl)-dimethylammonio!-2-hydroxy-1-propane sulfate
MES=2-N-morpholino-ethane sulfonic acid
Recovery of LDL (in percent) in dependency upon the VLDL contents mg/dl!
-□- without detergent
-x- 0.005% Zwittergent 3-14
The following examples explain the invention in greater detail:
Reagent solution 1
10 mM Bis-Tris Methane, pH 7,5
x% Zwittergent 3-14 (=N-tetradecyl-N,N-dimethylammonio-3-propane sulfonate)
Reagent solution 2
10 mM Bis-Tris Methane, pH 7,5
0.05 mg/ml PAMPS
An L DL-free VLDL preparation was first added to a native sample with a low VLDL contents (16 mg/dl). 250 μl of reagent solution 1 were then added to 5 μi of this sample and for 5 minutes incubated at 37° C. Subsequently, 50 μl of reagent solution 2 were added and incubated for another 5 minutes at 37° C. Immediately after this incubation, the resulting turbidity was measured bichromatically at 505 nm (reference wavelength 700 nm).
From Table 1 it can be seen that a concentration of 0.005 and 0.01% Zwittergent is effective; i.e. VLDL interference was almost completely eliminated while a significant interference was observed beginning with 25 mg/dl of VLDL when no detergent was added.
TABLE 1__________________________________________________________________________VLDL-Conc. LDL-Conc. (UZ) Zwittergent3-14(%); LDL values in mg/dlSample(mg/dl) (mg/dl) 0 0.0005 0.001 0.002 0.005 0.01__________________________________________________________________________1 16 118 143 140 147 141 136 1442 26 113 156 149 149 149 134 1343 44 103 170 176 179 171 130 1184 66 91 200 210 213 196 114 1125 88 79 214 206 213 202 73 81 % recovery1 16 118 121 118 125 120 115 1222 26 113 138 132 132 132 119 1193 44 103 165 171 174 166 126 1154 66 91 220 230 234 216 125 1235 88 79 271 261 270 256 92 102__________________________________________________________________________
cf. example 1
The following detergents were used in addition to Zwittergent 3-14
As described for example 1, an LDL-free VLDL preparation was added to a native sample with a low VLDL contents (16 mg/ml).
Table 2 shows that besides Zwittergent 3-14, Zwittergent 3-16 also shows a very good interference-reducing effect on the VLDL-spiked sample. Variants which contain a somewhat shorter alkyl group do not lead to an improvement in the concentrations tested.
TABLE 2__________________________________________________________________________ 1. Values in mg/dl ZwittergentVLDL-Conc. LDL-Conc. (UZ) without 3-14 Zwittergent 3-08Sample (mg/dl) (mg/dl) Zwittergent 0.005% 0.0025% 0.0050% 0.0075%__________________________________________________________________________1 16 118 136 137 136 140 1392 26 113 152 135 150 159 1603 44 103 181 130 181 189 1874 66 91 204 118 209 215 2155 88 79 226 92 226 228 233 2. % recovery1 16 118 116 116 115 119 1182 26 113 135 119 133 141 1423 44 103 176 126 176 184 1824 66 91 224 129 230 236 2365 88 79 287 117 286 289 294__________________________________________________________________________ 1. Values in mg/dl Zwittergent 3-10 Zwittergent 3-12 Zwittergent 3-16LDL-Conc. (UZ) 0.0025 0.0050 0.0075 0.0025 0.0050 0.0075 0.0025 0.0050 0.0075(mg/dl) % % % % % % % % %__________________________________________________________________________118 134 138 137 133 137 141 133 126 113113 156 145 156 150 159 165 140 132 106103 181 180 183 186 183 187 126 124 12391 207 209 207 207 213 214 125 126 12379 227 226 229 226 228 230 109 119 114 2. % recovery118 113 117 116 113 116 119 113 107 96113 138 129 138 133 141 146 124 117 93103 176 175 178 181 178 182 123 121 12091 228 229 227 227 234 236 137 138 13679 288 285 290 286 289 291 138 151 144__________________________________________________________________________
An LDL-free VLDL preparation with a VLDL contents of 64 mg/dl was tested as described in example 1. Since the preparation does not contain any LDL, the resulting LDL value must be 0 (optimal value).
______________________________________Reagent 1: 10 mM Bis-Tris Methane, pH 7,5 0.03 mg/ml PAMPS + detergentReagent 2: 10 mM Bis-Tris Methane, pH 7,5 120 mM MGCl2 + detergent______________________________________
TABLE 3______________________________________Detergent LDL (mg/dl)______________________________________without detergent 38.30.005% Zwittergent 3-14 1.00.01% Chaps 10.10.01% Chapso 10.00.01% Empigen 13.80.01% Phosphatidylcholin 11.80.01% Diheptadecanoyl 10.50.01% Dodecyldimethylaminoxide 8.2Phosphatidylcholin0.1% Pluronic F68/0.01% Tween 20 -0.6______________________________________
The effect of the detergent concentrations of the zwittergent types 3-10, 3-12, and 3-14 in native samples with varying VLDL contents was tested. The LDL and VLDL target values of the samples were determined using the UZ method.
______________________________________Reagent 1: 40 mM BIS-Tris Methane, pH 7,5 24 mM MGCl2 x% ZwittergentReagent 2: 40 mM Bis-Tris Methane, pH 7,5 0.15 mg/ml PAMPS x% Zwittergent______________________________________
Experimental design: cf. example 1
The results shown in Table 4 confirm that interference in samples containing up to 130 mg/dl VLDL can be completely eliminated. The use of Zwittergents with a shorter chain length x requires a higher concentration to reduce interference.
TABLE 4__________________________________________________________________________ Concentration measured (mg/dl) Sample 6 Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 LDL: 182 mg/dl LDL: 122 mg/dl LDL: 153 mg/dl LDL: 178 mg/dl LDL: 237 mg/dl LDL: 191 mg/dl VLDL:134 VLDL: 20 mg/dl VLDL: 35 mg/dl VLDL: 46 mg/dl VLDL: 46 mg/dl VLDL: 65 mg/dl mg/dl__________________________________________________________________________Zwittergent 3-100.001% 130 181 199 260 232 3140.005% 133 180 198 263 230 3130.025% 135 172 198 263 240 3130.05% 136 182 207 258 249 3180.25% 118 145 185 238 193 213Zwittergent 3-120.001% 133 190 205 252 248 3290.005% 134 186 206 253 238 3050.025% 134 177 188 237 195 2500.5% 122 154 179 235 189 190Zwittergent 3-140.001% 142 171 207 264 245 3100.005% 129 160 179 237 189 186 % recoveryZwittergent 3-100.001% 107 118 112 109 121 1730.005% 109 118 111 111 120 1720.025% 111 112 111 111 126 1720.05% 111 119 116 109 130 1750.25% 97 98 104 100 101 117Zwittergent 3-120.001% 109 124 115 106 130 1810.005% 110 121 116 107 125 1610.025% 110 116 106 100 102 1370.05% 100 101 101 99 99 104Zwittergent 3-140.001% 116 112 116 111 128 1700.005% 106 105 101 100 99 102__________________________________________________________________________
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|U.S. Classification||436/71, 436/825, 436/13, 436/815|
|Cooperative Classification||Y10T436/104165, Y10S436/815, Y10S436/825, G01N33/92, G01N2800/044|
|Feb 20, 1996||AS||Assignment|
Owner name: BOEHRINGER MANNHEIM GMBH, GERMANY
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:KARL, JOHANN;REEL/FRAME:007895/0543
Effective date: 19960212
|Jan 25, 2000||CC||Certificate of correction|
|Nov 21, 2000||CC||Certificate of correction|
|Feb 14, 2002||FPAY||Fee payment|
Year of fee payment: 4
|Mar 29, 2006||REMI||Maintenance fee reminder mailed|
|Sep 8, 2006||LAPS||Lapse for failure to pay maintenance fees|
|Nov 7, 2006||FP||Expired due to failure to pay maintenance fee|
Effective date: 20060908